This invention relates to mass spectrometers wherein the process of ionizing a sample involves the generation of a jet of charged particles. More particularly, it relates to mass spectrometers for the analysis of liquid samples by electrospray or atmospheric pressure ionization, but is also applicable to certain other types.
Complex mixtures of high molecular weight and/or thermally labile biomolecules are now routinely analyzed by electrospray or atmospheric pressure ionization mass spectrometry, often following separation by liquid chromatography or capillary electrophoresis. Most conveniently, to carry out such analyses the eluent from the chromatographic apparatus is fed directly to the electrospray or atmospheric pressure ionization source of a mass spectrometer. Both these ionization techniques are capable of generating intact molecular ions of very high molecular weight samples, and especially in the case of electrospray ionization, these ions may carry a large number of charges. This brings their mass-to-charge ratio into the range where it can be measured by relatively inexpensive mass analyzers such as quadrupoles or ion traps.
Both electrospray and atmospheric pressure ionization sources used for the analysis of solutions (rather than gases) involve the generation of a jet of charged particles in a region of high ambient pressure (typically atmospheric) and means for passing at least some of the charged particles into a region of lower pressure where they are mass analyzed. The jet typically comprises an aerosol of droplets produced from the solution, and the droplets may be at least partially desolvated by collisions with inert gas molecules in the region of high pressure.
In the case of an electrospray ionization source the aerosol is formed by maintaining a potential difference of between 3 and 6 kV between the exit of a capillary tube containing the solution to be analyzed and a counter electrode disposed downstream of it. The droplets comprised in the aerosol are electrically charged and are at least partially desolvated by collisions with molecules of an inert gas (usually heated) which is caused to flow through the region where the aerosol is formed. The charged particles so produced then pass through a nozzle-skimmer pressure reduction stage into an evacuated region where they are mass analyzed. In the case of many biomolecules the ions produced by electrospray ionization carry a large number of charges so that their mass-to-charge ratios may lie in the mass range of a quadrupole mass analyzer of moderate cost which could not be used if the ions were singly charged. Prior electrospray ionization sources are disclosed in U.S. Pat. Nos. 4,531,056, 4,542,293, 4,209,696, 4,977,320 and 5,504,327, PCT Patent Applications 95/24259, 98/11595 and 97/29508 and UK Patent Application 2,308,227. The technique of electrospray ionization has been reviewed by Fenn et. al. in Mass Spectrom. Rev. 1990, vol 9 pp 37-70 and Smith et. al. in Mass Spectrom. Rev. 1991 vol 10 pp 359-451.
Historically, API sources were developed for the analysis of trace materials in gases (for example, the source described in UK Patent 1,584,459), but are now extensively used for the analysis of liquids. In such a source the jet of charged particles is generated by first producing an aerosol of droplets from the solution by means of a nebulizer in a region of high ambient pressure, then charging the droplets by a separate ionization process, for example a corona discharge maintained between electrodes in the vicinity of the aerosol. Charged droplets so produced may be desolvated as in the case of an electrospray source and the charged particles so produced. See, for example, Homing, Carroll et al, J Chromatog. 1974 vol 99 pp 13-21. Instead of a corona discharge, other forms of ionization can be employed, for example a 63Ni radioactive foil, and many different types of nebulizer may be used. More recent ion sources based on these early devices are known as atmospheric pressure chemical ionization sources (APCI) because ionization is essentially a chemical ionization process, that is, the reaction of sample molecules with primary ions generated in the discharge or other means of primary ionization. In fact, a separate ionization process is not always required and in some ion sources the nebulization step itself generates a charged particle jet as a result of ion evaporation from the droplets which become electrically charged during their formation from the bulk liquid.
A further variation of API sources is known as thermospray ionization, in which the liquid is nebulized by causing it to flow through a strongly heated capillary tube. (See for example U.S. Pat. No. 4,730,111). This nebulization method often produces sufficient ions for subsequent mass analysis without an additional ionization step but may be assisted by a variety of methods such as a glow discharge or electron impact ionization. on sources that provide combinations of the features described, for use either imultaneously or as alternative nebulization or ionization methods in the same source, are also known. For example, most electrospray ion sources in current production also provide APCI capabilities. See, for example, Andrien and Boyle, Spectroscopy 1995 vol. 2 pp. 42-44, PCT patent applications 95/24259 and 98/11595 and GB patent application 2,308,227.
Charged particle jet sources of the types described are very frequently used to analyze the eluent from a liquid chromatograph, and are now employed in this way on a routine basis. The ability of these sources to interface directly to liquid chromatography and to produce characteristic ions from very high mass thermally labile molecules has recently created a demand for automated systems capable of analyzing many samples in as short a time as possible, for example in medical screening programs (for example, see Rasheed, Bucknall et al, Clin. Chem. 1997 vol 43:7 pp 1129-1141) and for DNA and protein sequencing (for example, PCT patent application 94/16101). Applications of this type require very high throughput if they are to be cost effective, but prior types of charged-particle source are capable of accepting the eluent of a single chromatograph only. An automatic flow-switching valve arrangement for a liquid chromatograph attached to an API source is taught by Hagiwara et al. (J. Mass Spectrom. Sec. Japan, 1996 vol 44 (2) pp 249-259) but this is intended to reduce contamination of the ion source during repeated analysis carried out by one chromatograph.
An electrospray ion source having several capillaries operating simultaneously is reported by Kostiainen and Brums (Rapid Commun. in Mass Spectrometry, 1994 vol 8 pp 549-58) but this apparatus is intended to improve ionization efficiency from a single flow of analyte. Andrien, Whitehouse, et. al, in PCT patent application WO 99/13492 (published Mar. 18, 1999) describe a multiple inlet electrospray/API mass spectrometer in which at least two of the solutions introduced are simultaneously ionized. The mixture of ions generated from the two solutions is then introduced into a mass analyzer. However, such simultaneous introduction inevitably results in mass spectral data that represents a mixture of the two solutions and the method is therefore limited in its applicability.
It is an object of the present invention, therefore, to provide a mass spectrometer comprising a charged-particle jet ionization source that is capable of receiving a plurality of fluid streams, each comprising a sample to be analyzed, without simultaneously introducing ions from more than one of the fluid streams into the mass analyzer of the spectrometer. It is another object of the invention to provide such a spectrometer that can produce mass spectral data from all of the streams quickly enough to allow the analysis of species in the streams that have been separated by high resolution liquid chromatography.
It is further object to provide methods of mass spectrometry using a charged-particle jet ionization source to analyze a plurality of fluid streams without simultaneously introducing ions from more than one of the streams into a mass analyzer. Another object of the invention is to provide such methods that are fast enough to allow the analysis of species in the fluid streams that have been separated by high resolution liquid chromatography. Further objects of the invention are to provide mass spectrometers comprising electro spray, thermo spray and/or APCI ion sources capable of analyzing more than one flow of fluid and methods of operating those mass spectrometers. It is yet another object to provide a liquid chromatograph mass spectrometer comprising a plurality of chromatographs which can be operated simultaneously and still another object to provide methods of operating it.
In accordance with these objectives the invention provides a mass spectrometer comprising an evacuated chamber, a sampling region where the pressure is greater than in said evacuated chamber, a sampling orifice communicating between said sampling region and said evacuated chamber, and a mass analyzer which receives at least some charged particles which pass along a first axis through said sampling orifice from said sampling region into said evacuated chamber, said spectrometer characterized by the further provision of:
a) a plurality of charged-particle jet generation means each of which is supplied with a fluid to be analyzed and generates a jet, aligned with a jet axis, of charged particles derived from said fluid, disposed so that each generation means has a different jet axis and so that all said jet axes intersect said first axis within said sampling region;
b) jet selecting means comprising a hollow member disposed so that the intersections of said jet axis and said first axis are within its interior, said hollow member having at least a first aperture alignable with said jet axis through which at least some charged particles comprised in a said charged-particle jet may enter the interior of said hollow member and travel to said first axis; and
c) means for aligning said aperture in said hollow member with each said jet axis in turn, thereby allowing in turn at least some charged particles comprised in each said jet to enter the interior of said hollow member to pass through said sampling orifice into said evacuated chamber, and subsequently to enter said mass analyzer.
In preferred embodiments at least a second aperture is provided in the hollow member of said jet-selecting means through which a charged particle jet entering through the first aperture may exit from the interior after intersecting the first axis. In this way the charged-particle jet suffers minimal disturbance when it is selected.
In a further preferred embodiment the invention provides an electrospray ionization mass spectrometer in which at least one said charged-particle jet generation means comprises an aerosol generation means maintained at a high potential relative to counter electrode means disposed downstream of it. Said counter electrode means may conveniently comprise said hollow member which may be made of an electrically conductive material, and/or an additional counter electrode disposed in the path of the jet of charged particles which exits from said hollow member when the apertures in it are aligned with the jet axis of the aerosol generation means. Said aerosol generation means may comprise a capillary tube. Preferably, a nebulizing gas is supplied to the exit of the capillary tube by means of a tube coaxial with the capillary tube to assist with the formation of the aerosol, as in prior types of single-jet electrospray sources. A heated drying gas may also be supplied to the exit of the aerosol generation means in said sampling region to assist desolvation of the droplets produced in the aerosol.
In an alternative embodiment an atmospheric pressure ionization mass spectrometer is provided, in which each charged-particle generation means comprises aerosol generation means for generating droplets from said fluid and means for electrically charging the droplets so produced. Aerosol heating means may also be provided for desolvating the droplets produced by the aerosol generating means. Conveniently the means for electrically charging the droplets may comprise a discharge electrode disposed in said sampling region and maintained at a potential which results in the formation of a corona discharge. The aerosol generating means may comprise a tube for supplying a nebulizing gas, as in the case of the electrospray jet generation means described above.
It is not necessary for all of the charged particle jet generation means to be identical. It is within the scope of the invention to use, for example, two electrospray ionization generation means and two atmospheric pressure ionization generation means. Further, one or more of the charged-particle jet generation means may comprise a thermospray ionization device, wherein ionization of the sample is effected by strongly heating a capillary tube through which the sample solution is flowing.
It is also advantageous to use one of the charged particle jet generation means to introduce a calibration compound for the mass spectrometer. In this way the mass spectrometer calibration may be updated during each sampling cycle and the mass measurement accuracy of the mass spectrometer consequently improved.
In further preferred embodiments the charged-particle jet generation means are radially disposed so that the jets they produce are directed towards said first axis, the means for aligning said aperture may comprise motor means for rotating said hollow member to bring an aperture in line with each of the charged-particle jet generation means in turn. Conveniently, the jets may be arranged to be perpendicular to the first axis, but this is not essential. This radial disposition allows at least some of the charged particles produced by each generation means to pass through the sampling orifice and be analyzed by the mass analyzer. If, as is preferred, more than one aperture is provided, these are typically arranged as diametrically opposed pairs so that in any given position of the hollow member where one aperture is aligned with one of the charged-particle generation means the other aperture of the pair provides an exit aperture through which the charged-particle jet may escape from the hollow member with minimal disturbance. In a preferred embodiment, two apertures are provided disposed directly opposite to one another and the hollow member is rotated to align the apertures in it with a particular jet axis.
The charged-particle jet generation means may be disposed around an arc of less than 180xc2x0 centered on said first axis. As the hollow member rotates, one aperture in it serves first to allow charged particles from each jet in turn to enter the hollow member while the other serves as a corresponding exit aperture. Further rotation of the hollow member then reverses the role of the two apertures so that the aperture previously serving as the exit aperture then becomes the entrance aperture during the next 180xc2x0 of rotation of the hollow member. It will be appreciated that more apertures can be provided if desired, providing that the arc around which the charged-particle jet generators are arranged is less than the angle between adjacent apertures in the hollow member.
Further preferably movement of the hollow member is stopped for a predetermined time period when an aperture in it is aligned with each of the charged-particle jet generation means in turn. This allows charged particles to be sampled from each generation means in turn for the predetermined time period without interference from the charged particles produced by the other generation means. Relevant data from the mass spectrometer is then acquired only while a particular charged particle jet is being sampled. Means may be produced for associating the data being acquired with the generation means being sampled at that time, for example by detecting the position of the jet selection means and flagging the corresponding data accordingly.
Apparatus according to the invention may also comprise a plurality of chromatographs, each feeding its eluent to a different charged-particle jet generation means. Typically, liquid chromatographs will be used in conjunction with electrospray ionization, but gas chromatographs or capillary electrophoresis separation devices may also be employed. Using four such chromatographs and their corresponding generation means disposed at 45xc2x0 to each other, a jet selection means comprising two apertures may sample each chromatograph eluent for 0.1 seconds and take 0.1 seconds to move between each of the generation means. Thus a complete mass spectrum may be generated for every chromatograph more frequently than once per second while four chromatographic analyses are being carried out.
Any convenient type of mass analyzer may be used in the invention, for example, magnetic sector, quadrupole, ion trap or time-of-flight analyzers , and tandem mass spectrometers such as triple-quadrupole mass spectrometers. Time-of-flight and ion-trap mass analyzers are especially appropriate because of their ability to substantially simultaneously detect ions of all mass-to-charge ratios. Consequently, an undistorted complete spectrum can be recorded in a shorter time while the jet selection means is sampling charged particles from any particular jet generation means than would be possible with a scanning mass analyzer. The data so acquired may then be processed in the time period when the jet selection means is moving between the generation means so that the analyzer is able to start acquisition as soon as the jet selection means is aligned with the next generation means. In this way it is possible to speed up the rate at which each of the jet generation means is sampled, thereby minimizing the loss of time-varying data from each of the generation means. However, tandem mass spectrometers, especially triple-quadrupole mass spectrometers, also benefit from the greater sample throughput that can be achieved using the present invention.
Viewed from another aspect the invention provides a method of mass spectrometry comprising mass analyzing charged particles which pass into an evacuated chamber through a sampling orifice along a first axis from a sampling region in which the pressure is greater than in the evacuated chamber, said method characterized by:
a)supplying a fluid to be analyzed to each of a plurality of charged-particle jet generator means to generate jets of charged particles derived from said fluid along a jet axis, each said charged-particle jet generation means having a different jet axis and each said jet axis intersecting said first axis within said sampling region; and
b) selecting in turn each of at least some of the jets of charged-particles by aligning with them a first aperture in a hollow member within whose interior said jet axis and said first axis intersect so that charged particles comprised in the jet so selected may enter the interior of said hollow body and travel to said first axis and at least some of said charged-particles pass along said first axis through said sampling orifice into said evacuated chamber.
A preferred method further comprises allowing said selected jet to exit through a second aperture in said hollow member. In other preferred methods the jet of charged particles is produced by electrospray ionization, for example by generating an aerosol of droplets at a high potential relative to counter electrode means disposed downstream of the charged-particle jet generation means. Alternatively, or in addition, the jet of charged particles may be produced by atmospheric pressure ionization, for example by generating an aerosol and electrically charging the droplets so produced by means of a corona discharge.
Further preferred methods according to the invention comprise repeating the cycle of mass analyzing in turn each of at least some of said jets of charged particles and acquiring mass spectral data for each selected jet of charged particles during a plurality of said cycles. In preferred methods the step of mass analyzing the charged particles comprises measuring their mass-to-charge ratios using a time-of-flight or an ion trap mass analyzer, but tandem mass analyzers, such as a triple-quadrupole can also be used. Still further preferred methods comprise selecting a said jet of charged particles for a predetermined time by maintaining said hollow member in a fixed position and acquiring mass spectral data for said predetermined time, then aligning said hollow member to select the next of said jets of charged particles for which mass spectral data acquired. inhibiting the acquisition of data while said alignment is taking place.
Typically, the fluid supplied to each of the charged-particle jet generation means comprises the eluent from a chromatograph. Thus the invention provides a method of carrying out simultaneously the mass spectral analysis of the eluent from a plurality of chromatographs without the need for a plurality of mass spectrometers or switching the eluent flows directly.